Cryopreservation is used to preserve biological samples over an extended period at ultra-low temperatures. This process evolved into vitrification, a more advanced and superior technology in which fluids or water molecules form a glass-like structure without forming ice crystals. Unlike fresh cells, cryopreservation is reported to reduce oocyte viability and developmental competency. This study employed two vitrification techniques, vitrification solution (VS) and Cryotop, to investigate the meiotic resumption in bovine. Oocytes were extracted from cow ovaries collected from slaughterhouses in Banting and Shah Alam, Selangor, Malaysia. The oocytes were grouped (A, B, and B’) based on cumulus morphology and matured in vitro in a culture dish (humidified 5% carbon dioxide incubator at 38.5°C) for 20 to 24 hr. Oocytes were vitrified after maturation using straws or aids of Cryotop sheets, then submerged in liquid nitrogen and stored for five days before defrosting for cryoprotectant elimination. By using Giemsa staining, the maturation state of fresh and vitrified bovine oocytes was evaluated through five parameters: zygotene, pachytene, diakinesis, metaphase I, and metaphase II. The maturation rate demonstrated only slight differences in the three groups of oocytes treated with VS (A: 44.79%; B: 30.97%; B’: 20.70%) and Cryotop (A: 39.42%; B: 37.27%; B’: 28.97%), which were significantly lower than fresh oocytes (A: 55.83%; B: 44.82%; B’: 56.17%). Both VS and Cryotop methods were viable options for cryopreserving oocytes, but the Cryotop technique was more effective in increasing the meiotic competence of poor-quality oocytes.

• Abd El-Aziz, A. H., Mahrous, U. E., Kamel, S. Z., & Sabek, A. A. (2016). Factors influencing in vitro production of bovine embryos: A review. Asian Journal of Animal and Veterinary Advances, 11(12), 737–756. https://doi.org/10.3923/ajava.2016.737.756

• Aguila, L., Treulen, F., Therrien, J., Felmer, R., Valdivia, M., & Smith, L. C. (2020). Oocyte selection for in vitro embryo production in bovine species: Noninvasive approaches for new challenges of oocyte competence. Animals, 10(12), 2196. https://doi.org/10.3390/ani10122196

• Aljaser, F. S. (2022). Cryopreservation methods and frontiers in the art of freezing life in animal models. In Y. Bozkurt & M. N. Bucak (Eds.), Animal reproduction. IntechOpen. https://doi.org/10.5772/intechopen.101750

• Amidi, F., Khodabandeh, Z., & Mogahi, M. H. N. (2018). Comparison of the effects of vitrification on gene expression of mature mouse oocytes using cryotop and open-pulled straw. International Journal of Fertility and Sterility, 12(1), 61–67. https://doi.org/10.22074/ijfs.2018.5112

• Angel-Velez, D., De Coster, T., Azari-Dolatabad, N., Fernandez-Montoro, A., Benedetti, C., Pascottini, O. B., Woelders, H., Soom, A. V., & Smits, K. (2021). New alternative mixtures of cryoprotectants for equine immature oocyte vitrification. Animals, 11(11), 3077. https://doi.org/10.3390/ani11113077

• Basirat, Z., Rad, H. A., Esmailzadeh, S., Jorsaraei, S. G. A., Hajian-Tilaki, K., Pasha, H., & Ghofrani, F. (2016). Comparison of pregnancy rate between fresh embryo transfers and frozen-thawed embryo transfers following ICSI treatment. International Journal of

• Reproductive Biomedicine, 14(1), 39–46.

• Bidin, H. (2005). IVF performance of different qualities of oocyte using co-culture and chemically-defined medium in Malaysian cattle [Unpublished Doctoral dissertation]. Universiti Malaya.

• Bidin, H., Osman, N. A., & Kamaruddin, M. (2012). Effects of oestradiol-17β on the development of cattle oocytes. In Proceedings of the 5th International Conference on Animal Nutrition 2012 Malacca, Malaysia (pp. 314-316). Malaysian Agricultural Research and Development Institute.

• Bottrel, M., Ortiz, I., Pereira, B., Díaz-Jiménez, M., Hidalgo, M., Consuegra, C., Morató, R., Mogas, T., & Dorado, J. (2019). Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos. Theriogenology, 125, 242–248. https://doi.org/10.1016/j.theriogenology.2018.11.011

• Budani, M. C., & Tiboni, G. M. (2020). Effects of supplementation with natural antioxidants on oocytes and preimplantation embryos. Antioxidants, 9(7), 612. https://doi.org/10.3390/antiox9070612

• Cobo, A., Bellver, J., Domingo, J., Pérez, S., Crespo, J., Pellicer, A., & Remohí, J. (2008). New options in assisted reproduction technology: The Cryotop method of oocyte vitrification. Reproductive Biomedicine Online, 17(1), 68–72. https://doi.org/10.1016/s1472-6483(10)60295-7

• Davachi, N. D., Shahneh, A. Z., Kohram, H., Zhandi, M., Dashti, S., Shamsi, H., & Moghadam, R. (2014). In vitro ovine embryo production: The study of seasonal and oocyte recovery method effects. Iranian Red Crescent Medical Journal, 16(9), e20749. https://doi.org/10.5812/ircmj.20749

• Dhali, A., Kolte, A. P., Mishra, A., Roy, S. C., & Bhatta, R. (2018). Cryopreservation of oocytes and embryos: Current status and opportunities. In D. S. Sheriff (Ed.), Infertility, assisted reproductive technologies and hormone assays. IntechOpen. https://doi.org/10.5772/intechopen.81653

• Díez, C., Muñoz, M., Caamaño, J. N., & Gómez, E. (2012). Cryopreservation of the bovine

• oocyte: Current status and perspectives.Reproduction in Domestic Animals, 47(s3), 76–83. https://doi.org/10.1111/j.1439-0531.2012.02029.x

• Do, V. H., Walton, S., Catt, S., & Taylor-Robinson, A. W. (2016). Requirements for cryopreservation of in vitro-produced bovine embryos by a standard method of vitrification. Journal of Veterinary Science and Animal Husbandry, 4(1), 102. https://doi.org/10.15744/2348-9790.4.102

• EXTOLSEED Oocyte and Sperm Bank (2018). Thawing (Kitazato). https://extolseed.com/vitrification-and-warming-kitazato/thawing-kitazato/

• Fathi, M., Moawad, A. R., & Badr, M. R. (2018). Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage. PLOS One, 13(3), e0194602. https://doi.org/10.1371/journal.pone.0194602

• Hajarian, H., Wahid, H., Rosnina, Y., Daliri, M., Dashtizad, M., Karamishabankareh, H., & Abas Mazni, O. (2011). Cryotop and development of vitrified immature bovine oocytes. Brazilian Journal of Veterinary and Animal Sciences, 63(1), 67–73. https://doi.org/10.1590/s0102-09352011000100011

• Hochi, S. (2022). Cryodevices developed for minimum volume cooling vitrification of bovine oocytes. Animal Science Journal, 93(1), e13683. https://doi.org/10.1111/asj.13683

• Idrissi, S. J., Le Bourhis, D., Lefevre, A., Emond, P., Le Berre, L., Desnoës, O., Joly, T., Buff, S., Maillard, V., Schibler, L., Salvetti, P., & Elis, S. (2021). Lipid profile of bovine grade 1 blastocysts produced either in vivo or in vitro before and after slow freezing process. Scientific Reports, 11, 11618. https://doi.org/10.1038/s41598-021-90870-8

• Iussig, B., Maggiulli, R., Fabozzi, G., Bertelle, S., Vaiarelli, A., Cimadomo, D., Ubaldi, F. M., & Rienzi, L. (2019). A brief history of oocyte cryopreservation: Arguments and facts. Acta Obstetricia et Gynecologica Scandinavica, 98(5), 550-558. https://doi.org/10.1111/aogs.13569

• Jain, J. K., & Paulson, R. J. (2006). Oocyte cryopreservation. Fertility and Sterility, 86(4), 1037–1046. https://doi.org/10.1016/j.fertnstert.2006.07.1478

• Kader, A. A., Choi, A., Orief, Y., & Agarwal, A. (2009). Factors affecting the outcome of human blastocyst vitrification. Reproductive Biology and Endocrinology, 7, 99. https://doi.org/10.1186/1477-7827-7-99

• Keshavarzi, S., Eftekhari, A. D., Vahabzadeh, H., Mehrafza, M., Taheripanah, R., Asgharnia, M., Esfandyari, S., Ghazifard, A., Hosseinirad, H., & Paktinat, S. (2022). A comparative study of post-warming survival rates and clinical outcomes of human blastocysts vitrified/warmed by CryoTouch and Cryotop methods. JBRA assisted Reproduction, 26(4), 568–573. https://doi.org/10.5935/1518-0557.20210116

• Kuwayama, M. (2007). Highly efficient vitrification for cryopreservation of human oocytes and embryos: The Cryotop method. Theriogenology, 67(1), 73–80. https://doi.org/10.1016/j.theriogenology.2006.09.014

• Kuwayama, M., Vajta, G., Kato, O., & Leibo, S. P. (2005). Highly efficient vitrification method for cryopreservation of human oocytes. Reproductive BioMedicine Online, 11(3), 300–308. https://doi.org/10.1016/s1472-6483(10)60837-1

• Liu, S., & Li, F. (2020). Cryopreservation of single-sperm: Where are we today? Reproductive Biology and Endocrinology, 18, 41. https://doi.org/10.1186/s12958-020-00607-x

• Magata, F., Ideta, A., Matsuda, F., Urakawa, M., & Oono, Y. (2021). Glutathione ethyl ester improved the age-induced decline in the developmental competence of bovine oocytes. Theriogenology, 167, 37–43. https://doi.org/10.1016/j.theriogenology.2021.03.004

• Mahmoud, K. G. M., El-Sokary, M. M. M., Kandiel, M. M. M., El-Roos, M. E. A. A., & Sosa, G. M. S. (2016). Effects of cysteamine during in vitro maturation on viability and meiotic competence of vitrified buffalo oocytes. Iranian Journal of Veterinary Research, 17(3), 165–170.

• Mogas, T. (2018). Update on the vitrification of bovine oocytes and in vitro-produced embryos. Reproduction, Fertility, and Development, 31(1), 105–117. https://doi.org/10.1071/rd18345

• Nagy, Z. P., Chang, C.-C., Shapiro, D., Bernal, D. P., Kort, H. I., & Vajta, G. (2009). The efficacy and safety of human oocyte vitrification. Seminars in Reproductive Medicine, 27(6), 450–455. https://doi.org/10.1055/s-0029-1241054

• Nagy, Z. P., Shapiro, D., & Chang, C.-C., (2020). Vitrification of the human embryo: A more efficient and safer in vitro fertilization treatment. Fertility and Sterility, 113(2), 241-247. https://doi.org/10.1016/j.fertnstert.2019.12.009

• Nikseresht, M., Toori, M. A., Rahimi, H. R., Fallahzadeh, A. R., Kahshani, I. R., Hashemi, S. F., Bahrami, S., & Mahmoudi, R. (2017). Effect of antioxidants (β-mercaptoethanol and cysteamine) on assisted reproductive technology in vitro. Journal of Clinical and Diagnostic Research: JCDR, 11(2), BC10–BC14. https://doi.org/10.7860/JCDR/2017/21778.9298

• Peinado, I., Moya, I., García-Valverde, L., Francés, R., Ribes, R., Polo, P., Gómez-Torres, M. J., & Monzó, A. (2022). Potential development of vitrified immature human oocytes: influence of the culture medium and the timing of vitrification. International Journal of Molecular Sciences, 24(1), 417. https://doi.org/10.3390/ijms24010417

• Prentice, J. R., & Anzar, M. (2010). Cryopreservation of mammalian oocyte for conservation of animal genetics. Veterinary Medicine International, 2011, 146405. https://doi.org/10.4061/2011/146405

• Rao, B. S., Mahesh, Y. U., Charan, K. V., Suman, K., Sekhar, N., & Shivaji, S. (2012). Effect of vitrification on meiotic maturation and expression of genes in immature goat cumulus oocyte complexes. Cryobiology, 64(3), 176–184. https://doi.org/10.1016/j.cryobiol.2012.01.005

• Reyes, J. N. V., & Jaramillo, L. C. (2016). Cryopreservation method and composition of the vitrification solution affect viability of in vitro bovine embryos. Revista Colombiana de Ciencias Pecuarias, 29, 130–137. https://doi.org/10.17533/udea.rccp.v29n2a06

• Rienzi, L., Romano, S., Albricci, L., Maggiulli, R., Capalbo, A., Baroni, E., Colamaria, S., Sapienza, F., & Ubaldi, F. (2010). Embryo development of fresh ‘versus’ vitrified metaphase II oocytes after ICSI: A prospective randomized sibling-oocyte study. Human Reproduction, 25(1), 66–73. https://doi.org/10.1093/humrep/dep346

• Rybska, M., Knap, S., Jankowski, M., Jeseta, M., Bukowska, D., Antosik, P., Nowicki, M., Zabel, M., Kempisty, B., & Jaśkowski, J. M. (2018). Cytoplasmic and nuclear maturation of oocytes in mammals – Living in the shadow of cells developmental capability. Medical Journal of Cell Biology, 6(1), 13–17. https://doi.org/10.2478/acb-2018-0003

• Sanches, B. V., Zangirolamo, A. F., & Seneda, M. M. (2019). Intensive use of IVF by large-scale dairy programs. Animal Reproduction, 16(3), 394–401. https://doi.org/10.21451/1984-3143-AR2019-0058

• Sasaki, H., Hamatani, T., Kamijo, S., Iwai, M., Kobanawa, M., Ogawa, S., Miyado, K., & Tanaka, M. (2019). Impact of oxidative stress on age-associated decline in oocyte developmental competence. Frontiers in Endocrinology, 10, 811. https://doi.org/10.3389/fendo.2019.00811

• Saunders, K. M., & Parks, J. E. (1999). Effects of cryopreservation procedures on the cytology and fertilization rate of in vitro-matured bovine oocytes. Biology of Reproduction, 6(1), 178–187. https://doi.org/10.1095/biolreprod61.1.178

• Sripunya, N., Somfai, T., Inaba, Y., Nagai, T., Imai, K., & Parnpai, R. (2010). A comparison of cryotop and solid surface vitrification methods for the cryopreservation of in vitro matured bovine oocytes. Journal of Reproduction and Development, 56(1), 176–181. https://doi.org/10.1262/jrd.09-108h

• Sydykov, B., Oldenhof, H., Sieme, H., & Wolkers, W. F. (2018). Storage stability of liposomes stored at elevated subzero temperatures in DMSO/sucrose mixtures. PLOS One, 13(7), e0199867. https://doi.org/10.1371/journal.pone.0199867

• Tao, T., & Del Valle, A. (2008). Human oocyte and ovarian tissue cryopreservation and its application. Journal of Assisted Reproduction and Genetics, 25, 287–296. https://doi.org/10.1007/s10815-008-9236-z

• Tharasanit, T., & Thuwanut, P. (2021). Oocyte cryopreservation in domestic animals and humans: Principles, techniques and updated outcomes. Animals, 11(10), 2949. https://doi.org/10.3390/ani11102949

• Tonev, I. D., Hristova, S. H., Zhivkov, A. M., & Mincheff, M. S. (2020). Cytotoxic effect of dimethyl sulfoxide (DMSO) on hematopoietic stem cells: Influence of the temperature and the incubation time. Bulgarian Chemical Communications, 52(Special Issue B), 40–43. https://doi.org/10.34049/bcc.52.B.0011

• Turathum, B., Gao, E.-M., & Chian, R.-C. (2021). The function of cumulus cells in oocyte growth and maturation and in subsequent ovulation and fertilization. Cells, 10(9), 2292. https://doi.org/10.3390/cells10092292

• Valdez, C. A., Abas Mazni, O., Takahashi, Y., Fujikawa, S., & Kanagawa, H. (1992). Successful cryopreservation of mouse blastocysts using a new vitrification solution. Journal of Reproduction and Fertility, 96(2), 793–802. https://doi.org/10.1530/jrf.0.0960793

• Vining, L. M., Zak, L. J., Harvey, S. C., & Harvey, K. E. (2021). The role of apoptosis in cryopreserved animal oocytes and embryos. Theriogenology, 173, 93–101. https://doi.org/10.1016/j.theriogenology.2021.07.017

• Vladimirov, I. K., Tacheva, D., & Dobrinov, V. (2019). The present and future of embryo cryopreservation. In B. Wu & H. L. Feng (Eds.), Embryology: Theory and practice. IntechOpen. https://doi.org/10.5772/intechopen.80587

• Whaley, D., Damyar, K., Witek, R. P., Mendoza, A., Alexander, M., & Lakey, J. R. T. (2021). Cryopreservation: An overview of principles and cell-specific considerations. Cell Transplantation, 30, 1-12. https://doi.org/10.1177/0963689721999617